One of the counting techniques used in flow cytometry relies on the measurement of the variation in impedance in a measuring zone when one or more particles are guided therethrough in-flow, this technique being referred to as the Coulter technique. The number of impedance changes indicates the number of particles whereas the measured impedance value is proportional to the volume of the particle. The counting method currently used in cytometry in conventional instruments is based on the detection of events of amplitude higher than a threshold. Each threshold crossing corresponds to one event. The Coulter technique may be used to count endogenous or exogenous particles such as cells, vesicles, parasites, beads, viruses, pollutants, or even toxins.
The main drawback of the principle of this counting technique is the lack of resolution in the presence of a high number of events. In this case, two or more events may coincide and not be distinguishable from one another. Specifically, two or more particles may pass more or less simultaneously through the measuring zone and thus interact together, this also having an effect on the measured impedance. When the number of particles to be counted greatly increases these coincidences become increasingly frequent, and detection may be limited by the resolution of the sensor.
In the presence of a very high number of events, the coincidence becomes such that the measured signal almost no longer drops below the threshold level. Thus, beyond a certain number, the increase in the frequency of the events translates into a decrease in the count. In order to prevent measurements that are completely corrupted by this saturation effect from being returned, an alarm system based on an indicator such as the occupation time of the sensor is provided in order to indicate the existence of a saturation of the sensor.
To compensate for events not detected because of coincidences, a correction of the count based on measurement of occupation time is possible.
Coincidence correction or linearization consists in extrapolating the number of real events from the counted events. The effect of this extrapolation increases with the number of events, having the consequence of increasing count uncertainty.
Linearization methods are based on extrapolation. They are therefore complex to implement, require a specific calibration for each machine produced, and pose verification problems.
The need to substantially correct the number of events counted increases with the number of particles to be counted, the result of this being to increase the uncertainty in the performed measurements. In the case where the sensor is saturated, the uncertainty in the result is too high to be corrected.
The invention aims to improve the situation. To this end, the invention provides a device for counting particles comprising a detector arranged to produce an electrical measurement signal in response to the passage of one or more particles, and a comparator arranged to compare the measurement signal with a threshold signal and to increment a counting value when the measurement signal exceeds the threshold signal. This device comprises a threshold-adjusting circuit that applies a lowpass filter to the measurement signal, and that is connected to the comparator in order to use the resulting signal as threshold signal.
This device is advantageous because the threshold signal is able to move as a function of the detected signal. Thus, when the detected signal increases because of the detection of many passages, the detection threshold also increases. This improves the resolution of the device and allows fewer counts to be lost.